Animal component free meningococcal polysaccharide fermentation and seedbank development

ABSTRACT

Animal-free meninge fermentation media and process is developed based upon use of a chemically defined medium. To improve polysaccharide production, fed-batch fermentation is examined using different feed solutions and feeding strategies. A feed solution containing glucose, amino acids, and trace metal elements produces Group A polysaccharide at approximately 3 times the level observed with batch fermentation. This process is used successfully to produce polysaccharides of  N. meningitidis  serotypes A, C, Y and W-135 and is run reproducibly at the 20L scale and can be scaled to 400L or more.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of vaccine preparation and, inparticular, fermentation of Neisseria bacteria, particularly N.meningitidis, for the production of polysaccharide for use in vaccines.

2. Summary of the Related Art

N. meningitidis causes both endemic and epidemic disease, principallymeningitis and meningococcemia. As a result of the control ofHaemophilus influenzae type b infections, N. meningitidis has become theleading cause of bacterial meningitis in children and young adults inthe United States (US), with an estimated 2,600 cases each year.(Recommendation of the Advisory Committee on Immunization Practices(ACIP). “Control and prevention of meningococcal disease and control andprevention of serogroup C meningococcal disease: evaluation andmanagement of suspected outbreaks.” MMWR 46: No. RR-5, 1997 6(hereinafter “ACIP”); CDC 1, Laboratory-based surveillance formeningococcal disease in selected areas—United States, 1989-1991, MMWR42: No SS-2, 1993 (hereinafter “CDC 1”).) The case-fatality rate is 13%for meningitis disease (defined as the isolation of N. meningitidis fromcerebrospinal fluid) and 11.5% for persons who have N. meningitidisisolated from blood (ACIP, CDC 1) despite therapy with antimicrobialagents (e.g., penicillin) to which US strains remain clinicallysensitive. (ACIP)

Based on multistate surveillance conducted during 1989 to 1991,serogroup B organisms accounted for 46% of all cases and serogroup C for45%; serogroups W-135 and Y and strains that could not be serotypedaccounted for most of the remaining cases. (ACIP, CDC 1) Recent dataindicate that the proportion of cases caused by serogroup Y strains isincreasing. (ACIP) In 1995, among the 30 states reporting supplementaldata on culture-confirmed cases of meningococcal disease, serogroup Yaccounted for 21% of cases. (CDC. Serogroup Y MeningococcalDisease—Illinois, Connecticut, and Selected Areas, United States,1989-1996. MMWR 46: Vol. 45, 1010-1013, 1996 (hereinafter “CDC 2”).)Serogroup A, which rarely causes disease in the US, is the most commoncause of epidemics in Africa and Asia. A statewide serogroup B epidemichas been reported in the US. (CDC. Serogroup B meningococcaldisease—Oregon 1994. MMWR 44: 121-124, 1995 (hereinafter “CDC 3”).)

N. meningitidis vaccines comprise group specific polysaccharideantigens. Several discoveries impacted the future of meningococcalpolysaccharide vaccines and demonstrated the significance ofanti-capsular antibodies in protection. (Frasch, “Meningococcalvaccines; past, present and future,” in Meningococcal Disease, ed. K.Cartwright. John Wiley and Sons Ltd, 1995.) In the late 1930s,serogroup-specific antigens of meningococcal serogroups A and C wereidentified as polysaccharides. (CDC 3) During the mid 1940s,investigators demonstrated that the protection of mice by anti-serogroupA meningococcal horse serum was directly related to its content ofanti-polysaccharide antibodies. (Frasch) Meningococcal polysaccharidevaccines were first demonstrated to be immunogenic in humans byGotschlich and his co-workers in the 1960s when immunization of US Armyrecruits with serogroup A and C polysaccharides induced protectiveantibodies. Id. The investigators recorded a significantly reducedacquisition rate of serogroup C carriage among vaccinated recruitscompared with unvaccinated individuals. Id.

Meningitidis polysaccharide manufacture requires fermentation of N.meningitidis. Current good manufacturing practice (cGMP) imposes severalcriteria to medium development for microbial fermentation for theproduction of biologics. Ideally, the medium should contain onlyessential components, be easily prepared in a reproducible manner, andsupport robust high-cell density culture. A chemically defined medium isinherently more reproducible than a complex medium. Furthermore, achemically defined medium enables discrete analysis of the effect ofeach component and strict control of medium formulation through identityand purity testing of raw materials. Finally, the fermentation mediumshould support the cultivation of the microorganism in question tohigh-cell density to improve volumetric productivity and to generate afinal culture whose composition and physiological condition is suitablefor downstream processing.

Catlin [J. Inf. Dis. 128:178-194, 1973], described a complex chemicallydefined medium named NEDF, containing approximately 54 ingredients,including all twenty naturally occurring amino acids, for growth ofNeisseria. In addition, Catlin described a medium called MCDA containing18 ingredients (in mM: NaCl, 100; KCl, 2.5; NH₄Cl, 7.5; Na₂HPO₄, 7.5;KH₂PO₄, 1.25; Na₃C₆H₅O₇.2H₂O, 2.2; MgSO₄.7H₂O, 2.5; MnSO₄.H₂O, 0.0075;L-glutamic acid, 8.0; L-arginine.HCl, 0.5; glycine, 2.0; L-serine, 0.2;L-cysteine HCl.H₂O, 0.06; sodium lactate, 6.25 mg of 60% syrup/mL ofmedium; glycerin, 0.5% (v/v); washed purified agar, 1% (wt/vol)CaCl₂.2H₂O, 0.25; Fe₂(SO₄)₃, 0.01) which was reported to support growthof Neisseria meningitidis on agar. The ability of MCDA to support growthin liquid medium (that is absent addition of agar) was not reported. LaScolea et al., [Applied Microbiology 28:70-76, 1974] reported on adefined minimal medium named GGM for the growth of Neisseriagonorrhoeae. The medium contained minimal salts, eight amino acids, twonitrogen bases, vitamins, coenzymes, metabolic intermediates andmiscellaneous components. La Scolea et al. reported growth of thisstrain to an optical density of 400 Klett units. An absorbance of 1 at600 nm is considered equivalent to 500 Klett units [see Gerhardt et al.,Manual of Methods for General Bacteriology, 1981, ASM., p. 197].Therefore, the maximum reported growth density achieved by LaScolea etal., was less than about one (1) absorbance unit. SU 1750689 A1described a method for preparing polysaccharide-protein vaccines againstNeisseria meningitidis B. A defined medium was described having thefollowing composition, g/L:

Sodium L-glutamate 1.30 ± 0.10 L-cysteine hydrochloride 0.03 ± 0.01Potassium chloride 0.09 ± 0.01 Sodium chloride 6.00 ± 1.00 Magnesiumsulfate heptahydrate 0.06 ± 0.01 Ammonium chloride 1.25 ± 0.01Disubstituted sodium phosphate 2.50 ± 0.20 dodecahydrate Trisubstitutedsodium citrate 0.50 ± 0.10 Glucose 1.60 ± 0.20

In this medium, it is reported that Neisseria may be cultured to a finaloptical density of 1.5±0.2 on the FEK-56M scale. This is an unfamiliarscale for optical density determination. However, based on the availablecarbon sources in the above noted medium, it is predictable that themaximum absorbance achievable would be in the range of about 1.5absorbance units.

U.S. Pat. No. 5,494,808 reports a large-scale, high-cell density (5 g/Ldry cell weight, and an optical density of between about 10-13 at 600nm) fermentation process for the cultivation of N. meningitidis. Thispatent disclose the following medium (called “MC.6”) for culturingNeisseria meningitidis for isolation of OMPC (“Outer Membrane ProteinComplex”) (all values in mg/L):

NaCl 5800 K₂HPO₄ 4000 NH₄Cl 1000 K₂SO₄ 1000 Glucose 10,000 L-GutamicAcid 3900 L-Arginine 150 Glycine 250 L-Serine 500 L-Cysteine•HCl 100MgCl₂•6H₂O 400 CaCl₂•2H₂O 28 Fe(III) Citrate 40

MENOMUNE® A/C/Y/W-135, Meningococcal Polysaccharide Vaccine, Groups A,C, Y and W-135 Combined, for subcutaneous use, is a freeze-driedpreparation of the group-specific polysaccharide antigens from Neisseriameningitidis, Group A, Group C, Group Y and Group W-135. N. meningitidisare cultivated with Mueller Hinton agar1 and Watson Scherp2 media. Thepurified polysaccharide is extracted from the Neisseria meningitidiscells and separated from the media by procedures which includecentrifugation, detergent precipitation, alcohol precipitation, solventor organic extraction and diafiltration.

SUMMARY OF THE INVENTION

Animal-free meninge fermentation media and process was developed basedupon use of a chemically defined medium. To improve polysaccharideproduction, fed-batch fermentation was examined using different feedsolutions and feeding strategies. A feed solution containing glucose,amino acids, and trace metal elements produces Group A polysaccharide atapproximately 3 times the level observed with batch fermentation. Thisprocess is successfully applied to serotypes A, C, Y and W-135. Thisprocess runs reproducibly at the 20 L scale and can be scaled to 400 Lor more.

The foregoing is summarizes certain embodiments of the invention (whichis more completely described below) and, therefore, should not beconstrued as limiting the invention in any manner. All patents, patentapplications, and other publications referred to in this specificationare hereby incorporated by reference in their entirety.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention comprises new compositions ofmatter for fermenting Nisseria. This composition is particularly usefulin fermenting Nisseria to produce a vaccine. The compositions of theinvention comprise aqueous compositions of matter comprising a solutionresulting from dissolving in water the compounds listed in one of thefollowing tables at the indicated concentrations (g/L)±10%:

TABLE 1a Modified Watson Scherp Medium I (MWSM I) Sodium phosphate,dibasic 2.500 Soy peptone 5-30 Monosodium Glutamate 5.000 PotassiumChloride 0.103 Magnesium sulfate 0.732 L-Cysteine 0.016 Glucose 11.250

TABLE 1b Modified Watson Scherp Medium II (MWSM II) Sodium phosphate,dibasic 2.500 Soy peptone 5-30 Monosodium Glutamate 5.000 PotassiumChloride 0.103 Magnesium sulfate 0.732 Glucose 11.250

TABLE 2a Meningitidis Chemically Defined Medium I (MCDM I) Glucose 10.00Soy Peptone 5-30 Sodium Chloride 5.80 Potassium Sulfate 1.00 PotassiumPhosphate, dibasic 4.00 L-Glutamic Acid 5.00 L-Arginine 0.30 L-Serine0.50 L-Cysteine 0.23 Magnesium Chloride 0.19 Calcium chloride 0.021Ferrous Sulfate 0.002

TABLE 2b Meningitidis Chemically Defined Medium II (MCDM II) Glucose10.00 Soy Peptone 5-30 Sodium Chloride 5.80 Potassium Sulfate 1.00Potassium Phosphate, dibasic 4.00 Magnesium Chloride 0.19 Calciumchloride 0.021 Ferrous Sulfate 0.002

We have surprisingly found that NH₄Cl (employed in prior art media) isnot readily consumed during Nisseria fermentation and is possibly evendeliterious. In some experiments, polysaccharide yield is roughly 20-50%greater when NH₄Cl is omitted from the media. Accordingly, we omit thiscomponent and surprisingly find that polysaccharide yield is improved.Therefore, the present invention provides a fermentation compositionwherein the composition omits NH₄Cl, and an improved method offermenting Nisseria in a fermentation composition wherein thecomposition omits NH₄Cl.

More preferably, however, the ammonium chloride nitrogen source isreplaced with a soy peptone as a nitrogen source. As known by thoseskilled in the art, soy peptone is enzymatically hydrolyzed soy refinedto remove impurities. Preferably 5-30 g/L of soy peptone is used. Morepreferably, 1-15 g/L is used in the fermentation composition. Among thesoy peptone's that can be used in the compositions of the presentinvention are SESOMAF-UF, Freetone A-1, HSP-A, and HY Soy UF. In onepreferred embodiment, the soy peptone is HSP-A (Nutricepts, Inc.;Minneapolis, Minn.). HSP-A has the following composition:

TABLE 4 Soy Peptone Composition Flowable spray dried powder Yes ColorLight Tan Protein 51%  Amino Nitrogen 3% Total Nitrogen 8% AN/TN ratio .38 Ash <10%  Moisture <8%  pH 6.5 Sodium 1% Potassium 4%

TABLE 5 Amino Acid Profile (mg/g) of Soy Peptone Amino Acid Free TotalASP 6 45 SER 9 30 GLU 15 85 GLY 2 20 HIS 6 15 ARG 14 40 THR 5 20 ALA 520 PRO 3 25 CYS NA 5 TYR 5 15 VAL 8 20 MET 4 5 LYS 16 30 ILE 9 20 LEU 1930 PHE 11 20 TOTAL 137 445

MCDM I differs from prior art MCDM in that a soy peptone replaces NH₄Clas a nitrogen source. MCDM II differs from MCDM I in that the aminoacids (other than those contributed by the soy peptone) have beenremoved from the composition; it is expected that the amino acidssupplied by the soy peptone are sufficient to sustain Nisseria growth.

Similarly, MWSM I differs from prior art MWSM in that a soy peptonereplaces NH₄Cl as a nitrogen source. MWSM II differs from MWSM I in thatthe amino acids (other than those contributed by the soy peptone) havebeen removed from the composition; it is expected that the amino acidssupplied by the soy peptone are sufficient to sustain Nisseria growth.

The components of the foregoing compositions are commercially availableand the compositions can be routinely made by simply dissolving thecomponents in water.

As mentioned, the compositions according to the invention are useful forNisseria fermentation, especially for the production of vaccines,particularly vaccines comprised of Nisseria polysaccharides, and moreparticularly of Nisseria polysaccharides of serotypes A, C, Y and W135,e.g., MENOMUNE®.

In another aspect, the invention comprises a method of fermentingNisseria in animal-free media. Any of the media of the invention can beemployed. As used herein, the term Animal-Free Nisseria Medium (“AFNM”)refers to any of MWSM I, MWSM II, MCDM I, and MCDM II. In oneembodiment, the method comprises (a) fermenting Neisseria in AFNM on oneor more seed stages followed by (b) fermenting Neisseria in AFNM as thebase medium and feed solution. Preferably, MCDM I is the medium used inall stages of the method. Preferably, the scale of each subsequentfermentation in the method is larger than the previous fermentation.

The parameters employed in the method of the invention (e.g., number ofseed stages, level of growth at which fermentation is moved from onefermentor to the next, feed rate of feed solution, etc.) are dependenton a number of factors, including the growth characteristics of thestrain and batch of Nisseria used (which will vary from strain to strainand batch to batch), the type of equipment employed, work schedules,etc. Suitable parameters include those provided in this specificationbut may vary significantly. Nevertheless, the state of the art is suchthat it would require no more than routine experimentation for one ofordinary skill in the fermentation art to determine suitablefermentation parameters useful and, indeed, optimal in the method of theinvention under the particular circumstances the artisan finds himself.

In one embodiment, the method comprises:

-   -   inoculating a vial (e.g., 1 ml) of Neisseria to a first flask        (e.g., 1 L) containing AFNM medium (e.g., 220 ml);    -   cultivating the flask (e.g., in a shaker at 36±1° C., 250 rpm        for 4-8 hours) to form a seed culture;    -   transferring (e.g., at OD of about 2) seed culture (e.g., about        10%) to one or a plurality of second flasks (e.g., three 2.8 L        flasks) containing AFNM (e.g., 700 ml);    -   fermenting the contents of the second flask(s) (e.g., at pH        6.8±0.2, temperature 36±1° C., DO 30%, airflow at constant 15        L/min; 2.5M phosphoric acid and 2.5M sodium hydroxide can be        used for pH control and 300% Dow 1520 antifoam solution to        control foaming);    -   transferring the contents of the second flask(s) (e.g., at OD        between 3-6) aseptically to a fed-batch fermentor (e.g., 400 L        fed-batch fermentor) where AFNM is the fermentation base medium        (e.g., at pH 6.8±0.2, temperature 36±1° C., DO 30%, with        agitation 250-270 rpm,    -   airflow gradually increase to maximum 300 L/min and then        gradually increasing back pressure to 8-12 psi to maintain DO);        and    -   feeding AFNM solution into the fermentor (e.g., when glutamate        reaches about 2 g/L),    -   preferably at rate of 5.6 L/hr for first 2 hours feeding and        then increase to 7.8 L/hr.

In further aspect, the invention comprises a method of producingNeisseria polysaccharide comprising fermenting Neisseria according tothe any of the methods described above and harvesting thepolysaccharide. Typical harvest is done when hourly increase in OD slowsand growth reaches stationary phase. Methods of harvesting Neisseriapolysaccharide are known to those skilled in the art. In a preferredembodiment, the use of a fed-batch fermentor, wherein some or allnutrients are supplied continuously or intermittently and all productsharvested at the end of fermentation, results in a significant increasein polysaccharide production.

The following Examples are provided for illustrative purposes only andare not intended to limit the invention in any manner. Those skilled inthe art will recognize that variations and modifications of thefollowing Examples may be employed without deviating from the spirit orliteral scope of the invention.

EXAMPLES

Unless otherwise indicated, the composition of the MCDM used in thefollowing experiments was the same as MCDM I except that 1 g/L of NH₄Clwas used in place of soy peptone.

Example 1 Fed Batch Animal-Free Fermentation Process Development

Fed-batch fermentation is examined using various feed solutions andfeeding under different growth conditions. Fed-batch fermentationproduces much higher polysaccharide levels than batch fermentation. Itis found that glucose residual remained high at the end of fermentationin subsequent fed-batch fermentations when 200 g/L of glucose is used inthe feed solution. Therefore, 100 g/L and 50 g/L of glucose in feedsolutions are compared. When 50 g/L of glucose is used, low glucoseresidual is obtained at end of fed-batch fermentation whilepolysaccharide remains relatively unchanged. Thus, 50 g/L of glucoseconcentration is used in the feed solution. Final feed solutioncomponents are listed in Table 6.

TABLE 6 Feed Solution Components (g/L) Glucose 50 Glutamic acid 50Arginine 3 Serine 3 Cysteine 2 NH₄Cl 10 MgCl₂ 2 CaCl₂ 0.14 FeSO₄ 0.02

Example 2 Animal-Free Medium and Process Improvement: Poor Utilizationof Ammonium Ion

It is noticed that ammonium ion residual remained relatively constantdue to minimal consumption. 2-L fermentations are carried out in orderto examine the effect of NH₄Cl on both polysaccharide production andcell growth in either the base medium and/or feed solution. Table 7lists an average of maximum OD₆₀₀ and polysaccharide from duplicatefermentations for each condition. Higher levels of PS are observed whenNH₄Cl is removed from both fermentation medium and feed solution. Asimilar result is observed at the 400-L scale. Elimination of NH₄Cl fromboth the base medium and feed solution improves polysaccharide yield andgrowth compared to inclusion of ammonium only in the base medium. Bothmaximum polysaccharide (393 mg/L) and growth (OD 5.5) without NH₄Cl inthe medium are higher than with NH₄Cl in the medium (PS 269 mg/L and OD4.5).

TABLE 7 Effect of NH₄Cl in MCDM^(†) and/or feed solution on growth andpolysaccharide production at 2L batch fermentation for group C (079C72)*Max. *Max. PS NH₄Cl OD (mg/L) Base MCDM & Feed 9.1 377 Base MCDM only8.1 403 No NH₄Cl 7.9 447 Average of duplicate experiments

Example 3 Nitrogen Source Screen in Watson Scherp Medium

Since inorganic nitrogen as NH₄Cl is removed, the effect of alternativesoy-based organic nitrogen sources on growth and polysaccharideproduction is examined. Experiments are performed with Watson Scherpmedium, the current manufacturing standard, and nitrogen sourcesFreetone A-1, HSP-A, SE50MAF-UF are selected for study. Testing is donein shake flasks and 2-L batch fermentations with Watson Scherp medium,in which casamino acids are replaced on a nitrogen content basis, byeach soy-based nitrogen source as shown in Table 8. Table 9 listsaverage maximum OD and polysaccharide from duplicate fermentations foreach condition Average maximum OD 7.9 and PS 468 mg/L are obtained withFreetone A-1; average maximum OD 11.2 and PS 510 mg/L with HSP-A; andaverage maximum OD 7.8 and PS 491 mg/L with SE50MAF-UF. These resultsshow polysaccharide yield from both HSP-A and SESOMAF-UF is higher thanthat from Freetone A-1. Therefore HSP-A and SE50MAF-UF are chosen forfurther testing.

TABLE 8 Watson Scherp with different organic nitrogen sources (g/L)Sodium phosphate, dibasic 2.500 Freetone A-1/SE50MAF-UF/HSP-A16.76/24.96/27.8 Monosodium Glutamate 5.000 Potassium Chloride 0.103Magnesium sulfate, crystals 0.732 L-Cysteine HCl Monohydrate 0.023Glucose 11.250

TABLE 9 Effect of nitrogen source on growth and Polysaccharideproduction 2L scale batch fermentation for group Y Ave. Max. Ave. Max.PS Nitrogen OD (mg/L) Freetone A-1 7.9 468 HSP-A 11.2 510 SE50MAF-UF 7.8491

A similar batch fermentation experiment is performed in which the twobest nitrogen sources from the previous work are compared to the currentnitrogen source standard, HY Soy UF. Table 10 lists average maximum ODand polysaccharide from duplicate fermentations for each condition.Maximum OD 7.0 and PS 378 mg/L are obtained with HY Soy UF; averagemaximum OD 9.5 and PS 602 mg/L with HSP-A; and, average maximum OD 7.8and PS 595 mg/L with SE50MAF-UF. Fermentation results show that bothcell growth and polysaccharide yield from both HSPA and SESOMAF-UF ishigher than that from HY Soy UF.

TABLE 10 Effect of nitrogen on growth and polysaccharide production 2Lscale batch fermentation for group Y (079C165) Ave. Max. Nitrogen ODAve. Max. PS (mg/L) HY SOY* 7.0 378 HSP-A 9.5 602 SE50MAF- 7.8 595 UFData from one fermentation

Interestingly, glucose and glutamate are utilized to exhaustion in thosefermentations containing HSP-A. Previous work with all meningitidisserogroups and MCDM type media result in variable growth andpolysaccharide production. One characteristic of those fermentations isvariable and incomplete utilization of glucose and glutamate substrates.To our surprise, fermentations containing HSP-A as the nitrogen sourcetotally consume the glucose and glutamate and as a likely outcomeresulted in higher levels of both cell growth and polysaccharideproduction. This characteristic has been shown to be highly reproducibleat both 2-L and 300-L scale fermentations. Other nitrogen sources do notexhibit this characteristic. Thus, HSP-A is the preferred nitrogensource.

Example 4 MCDM/HSP-A Development

It is found that HSP-A nitrogen source promotes the best growth andstimulates the highest polysaccharide production in Watson Scherpmedium. Since NH₄Cl is shown to cause variable results with respect togrowth and polysaccharide production in minimal chemically definedmedium (MCDM), the decision is made to substitute HSP-A for ammonium ona nitrogen basis in that medium. In that way it could be determinedwhether an organic nitrogen source was more acceptable for growth and/orpolysaccharide production in Neisseria meningitidis. Both MCDM andMCDM/HY Soy, in which HY Soy replaces NH₄Cl on a nitrogen basis, areused as controls. 2×2 L fermentations for each condition are performed.Table 11 lists average maximum OD and polysaccharide from duplicatefermentations for each condition. Average maximum OD 6.4 and PS 234 mg/Lwith MCDM; average maximum OD 6.7 and PS 199 mg/L with MCDM/HY Soy; andaverage maximum OD 7.1 and PS 288 mg/L with MCDM/HSP-A are obtained.These results show that MCDM/HSP-A resulted in the best polysaccharideyield and supported the highest growth.

TABLE 11 Effect of nitrogen source on growth and polysaccharideproduction 2L scale batch fermentation for group Y (079C191) Ave. Max.Ave. Max. PS Nitrogen OD (mg/L) MCDM 6.4 234 MCDM/HY 6.7 199 SoyMCDM/HSP-A 7.1 288

HSP-A concentration is varied in MCDM medium in order to examine theeffect of HSP-A concentration on growth and polysaccharide production.Table 12 lists average maximum OD and polysaccharide from duplicatefermentations for each condition. Average maximum OD was 6.4 and PS 226mg/L with 3.2 g/L of HSP-A; average maximum OD 10.4 and PS 346 mg/L with10 g/L of HSP-A; and average maximum OD 11.4 and PS 317 g/L with 28 g/LHSP-A. These results indicate that 10 g/L of HSP-A maximizedpolysaccharide production. Therefore, 10 g/L of HSP-A is used in MCDMmedium for further experimentation.

TABLE 12 Effect of HSP-A concentration on growth and polysaccharideproduction at 2L fermentation scale for group Y (087C4) Ave. Max. Ave.Max. PS HSP-A conc. OD (mg/L) 3.2 g/L  6.4 226 10 g/L 10.4 346 28 g/L11.4 317

To examine whether MCDM/HSP-A is suitable for other serotypes, 2 Lfermentations for group A, C, W135 and Y are performed. Table 13 listsaverage maximum OD and polysaccharide from duplicate fermentations foreach serotype except for group Y, for which only a single fermentationis performed. Average maximum OD 11.1 and PS 745 mg/L are obtained forgroup A; average maximum OD 10.4 and PS 453 mg/L for group C; and,average maximum OD 11.2 and PS 684 mg/L for group WI 35. For group Y,maximum OD 12.5 and PS 466 mg/L are observed in a single fermentation.These results show that MCDM/HSP-A is suitable for growth andpolysaccharide production by all 4 serotypes. Since all serogroupsexhibit similar behavior in MCDM/HSP-A medium (Table 13), it is feltthat a single serogroup could be used for sets of experiments targetingprocess improvement, and likewise that those serogroups could be usedinterchangeably between sets of experiments, as subsequentlydemonstrated.

TABLE 13 Application of MCDM/HSP-A to all four serotypes A, C, W135 andY at 2L fermentation scale (087C23) Ave. Max. PS Serotype Ave. Max. OD(mg/L) A 11.1 745 C 10.4 453 W135 11.2 684 Y* 12.5 466 *For group Y,only one fermentor was run.

Example 5 Fed-Batch Fermentation with MCDM/HSP-A

To further increase polysaccharide yield, fed-batch fermentation isexamined. Glutamate concentration is increased to 6 g/L from 5 g/L sinceit is observed that glutamate is exhausted earlier than glucose duringthe fermentation. Table 14 lists average maximum OD and polysaccharidefrom duplicate fermentations for each condition with Serogroup A.Average maximum OD 11.0 and PS 1075 are obtained by batch fermentation.Average maximum OD 14.2 and PS 1424 mg/L are observed with fed-batchfermentation with MCDM feed solution 5 as listed in Table 15. And,average maximum OD 19.5 and PS 1330 mg/L are obtained for fed-batchfermentation with HSP-A feed solution 1, as listed in Table 16. Theseresults show that fed-batch fermentation with MCDM feed solutionproduces the best polysaccharide yield and also supports very highgrowth. Final specific product yields (i.e., maximum yield divided bymaximum OD) for batch, MCDM feed and HSP-A feed are 97.7, 100.3 and68.2, respectively.

TABLE 14 Effect of fed-batch fermentation on growth and polysaccharideproduction at 2L scale for group A (087C43) Ave. Max. Ave. Max. PSSpecific Yield Fermentation OD (mg/L) (mg/L · OD) Batch 11.0 1075 97.7MCDM 14.2 1424 100.3 Feed HSP-A 19.5 1330 68.2 Feed

TABLE 15 MCDM feed solution components Dextrose 75.00 g/L MonosodiumGlutamate 37.500 g/L L-Arginine Monohydrate 3.00 g/L L-Serine 3.00 g/LL-Cysteine 2.00 g/L Magnesium Chloride•6H2O 2.00 g/L Calcium ChlorideDihydrate 0.15 g/L Ferrous Sulfate•7Hydrate 0.02 g/L

TABLE 16 HSP-A/Watson Scherp feed solution components Dextrose 75.00 g/LHSP-A 185.00 g/L Ferrous Sulfate 0.0468 g/L Potassium Chloride 0.75 g/LL-Cysteine HCl Monohydrate 0.45 g/L Monosodium Glutamate 37.50 g/L

For group C experiments two feed regimes, MCDM feed solution or MCDMfeed supplemented with HSP-A (as indicated in Table 17) are compared. Inorder to match the glucose and glutamate consumption rates observed inprevious fermentations, MCDM feed 5 components are increased 1.5-fold inthe feed solution. As shown in Table 18, average maximum OD 15.4 and PS560 mg/L are obtained by batch fermentation; average maximum OD 23 andPS 926 mg/by fed-batch fermentation with MCDM feed solution 6; andaverage maximum OD 30.7 and PS 908 mg/L by fed-batch fermentation withMCDM/HSP-A feed solution. These results indicate that fed-batchfermentation with MCDM feed solution produces the highest polysaccharideyield and also provides the highest PS specific production. Thepolysaccharide yield from fed-batch fermentation is much higher thanthat from batch fermentation for both groups A (previous experiment) andC.

TABLE 17 MCDM feed solution components Dextrose 112.5 g/L MonosodiumGlutamate 56.25 g/L L-Arginine Monohydrate 4.50 g/L L-Serine 4.50 g/LL-Cysteine 3.00 g/L Magnesium Chloride•6H2O 3.00 g/L Calcium ChlorideDihydrate 0.23 g/L Ferrous Sulfate•7Hydrate 0.03 g/L HSP-A (supplementexperiment) 90.00 g/L

TABLE 18 Effect of fed-batch fermentation on growth and polysaccharideproduction at 2L scale for group C (087C76) Ave. Max. Ave. Max. PSSpecific yield Fermentation OD (mg/L) (mg/L · OD Batch 15.4 560 36.4MCDM Feed 23.0 926 40.3 HSP-A Feed 30.7 726 23.6

Example 6 Scale-Up of Animal-Free Fermentation Process to 300-L

To examine whether the animal component free fermentation process isscalable, 300-L batch fermentation is performed with MCDM/HSP-A. 4×1-mLvials from the Product Development Working Seed Bank (WSB) areinoculated into 220 ml WS/HSP-A/Glut in 1 L shake flask as listed inTable 19. When OD reaches about 2, seed cultures are transferred tosecond stage 3×2.8 L shake flasks, each containing 700 ml WS/HSP-A/Glut.At OD between 1.2 and 1.6, a 10% inoculum is used to inoculate seedculture from shake flask to 30 L fermentor with 20 L WS/HSP-A/Glutmedium. Fermentation is controlled at pH 6.8±0.2, temperature 36±1° C.,DO 30%, airflow at constant 15 L/min. At OD between 3-6, the 20 L seedculture is transferred to the 300-L fermentor.

TABLE 19 WS/HSP-A/Glut medium components Sodium phosphate, dibasic 2.500g/L HSP-A 27.800 g/L Monosodium Glutamate 5.000 g/l Potassium Chloride0.103 g/L Magnesium sulfate, crystals 0.732 g/L L-Cysteine HClMonohydrate 0.023 g/L Dextrose 11.250 g/L

300-L batch fermentation is controlled at pH 6.8±0.2, temperature 36±1°C., DO 30%. Control parameters are cascaded to maintain DO at 30%;agitation gradually increased to 280 rpm from 100; airflow graduallyincreased to 300 L/min from 75 L/min, and finally back pressure isgradually increased to 8 psi from 4 psi. If necessary, agitation isfurther gradually increased to maximum 500 rpm. The fermentation isharvested when hourly increase in OD slowed, indicating growth hadreached stationary phase.

Table 20 lists seed culture OD and time for different seedtrain stagesfor serogroups A, C, and Y. It takes approximately 44.5 hours to attaintransfer OD of about 2 in the first stage seed shake flask withWS/HSP-A/Glut medium; 1.75-2.5 hours to reach transfer OD ofapproximately 1.2 in the second stage flask; and 3-4 hours to attain atransfer OD of 3 in the 30-L fermentor. Table 21 summarizes the resultsfrom three 300-L runs, one each for groups A, C, and Y. Maximum OD 10.3and PS 441 mg/L are observed for lot 085C22 group Y; maximum OD 10.2 andPS 653 mg/L for lot 087C42 group A; and maximum OD 8.3 and PS 272 mg/Lfor group C lot 087C103.

TABLE 20 Seed train OD and time N. meningtidis 1st Shake Flask 2^(nd)Shake Flask 20L Seed Vessel Lot No. Sero-type Hours OD Hours OD Hours OD087C22 Y 4.5 2.09 2.0 1.24 3.0 2.78 087C112 W-135 4.5 2.03 2 1.15 3.752.63 087C129 C 4 2.45 1.75 1.51 3.75 3.01 087C137 A 4.5 2.18 2.75 1.353.25 2.66

TABLE 21 400 L fermentation OD and PS summary Max. OD/ Max. PS Lot No.Serotype Hr (mg/L)/Hr 087C22 Y 10.3/6 441/7 087C112 W-135 10.2/7 650/7087C129 C  8.5/6 424/6 087C137 A 11.8/7 456/7

The embodiments provided herein are intended to illustrate specificembodiments of the present invention and are not intended to limit thescope of the invention. It is understood that alternative sources ofsalts, amino acids and the like may be used to substitute specificcomponents described herein.

1. A fermentation composition, comprising compounds in a ratio, byweight, of 2.5±10% sodium phosphate, dibasic, 5-30±10% soy peptone,5±10% monosodium glutamate, 0.103±10% potassium chloride, 0.732±10%magnesium sulfate, 0.016±10% L-Cysteine, and 11.250±10% glucose, andwherein the composition does not comprise NH₄Cl.
 2. The compositionaccording to claim 1, wherein the composition is aqueous.
 3. Thecomposition according to claim 1, wherein the soy peptone is HSP-A®. 4.A fermentation composition, comprising compounds in a ratio, by weight,of 10±10% glucose, 5-30±10% soy peptone, 5.8±10% Sodium Chloride, 1±10%potassium sulfate, 4±10% potassium phosphate, dibasic, 5-6±10%L-Glutamic Acid, 0.3±10% L-Arginine, 0.5±10% L-Serine, 0.23±10%L-Cysteine, 0.19±10% Magnesium Chloride, 0.021±10% Calcium chloride, and0.002±10% Ferrous Sulfate, and wherein the composition does not compriseNH₄Cl.
 5. The composition according to claim 4, wherein the compositionis aqueous.
 6. The composition according to claim 4, wherein the soypeptone is HSP-A®.
 7. A fermentation composition, comprising compoundsin a ratio, by weight, of 10±10% glucose, 5-30±10% soy peptone, 5.8±10%Sodium Chloride, 1±10% potassium sulfate, 4±10% potassium phosphate,dibasic, 0.19±10% Magnesium Chloride, 0.021±10% Calcium chloride, and0.002±10% Ferrous Sulfate, and wherein the composition does not compriseNH₄Cl.
 8. The composition according to claim 7, wherein the compositionis aqueous.
 9. The composition according to claim 7, wherein the soypeptone is HSP-A®.
 10. A method of fermenting Neisseria, comprisingfermenting Neisseria in a fermentation composition wherein thefermentation composition does not comprise NH₄Cl.
 11. The methodaccording to claim 10, wherein the fermentation composition is accordingto claim
 1. 12. The method according to claim 10, wherein thefermentation composition is according to claim
 4. 13. The methodaccording to claim 10, wherein the fermentation composition is accordingto claim
 7. 14. A method of fermenting Neisseria, wherein the Neisseriaare fermented in multiple batches wherein at least one fermentation isin a fermentation composition of claim
 1. 15. A method of fermentingNeisseria, wherein the Neisseria are fermented in multiple batcheswherein at least one fermentation is in a fermentation composition ofclaim
 4. 16. A method of fermenting Neisseria, wherein the Neisseria arefermented in multiple batches wherein at least one fermentation is in afermentation composition of claim
 7. 17. A method of fermentingNeisseria, comprising: inoculating a vial of Neisseria to a first flaskcontaining a fermentation composition; cultivating the Neisseria in thefirst flask; transferring the Neisseria from the first flask to aplurality of second flasks containing a fermentation composition;fermenting the contents of the second flasks; transferring the contentsof the second flasks to a fed-batch fermentor containing a fermentationcomposition; fermenting the contents of the fed-batch fermentor with afermentation composition, wherein at least one fermentation compositionis of claim
 1. 18. A method of fermenting Neisseria, comprising:inoculating a vial of Neisseria to a first flask containing afermentation composition; cultivating the Neisseria in the first flask;transferring the Neisseria from the first flask to a plurality of secondflasks containing a fermentation composition; fermenting the contents ofthe second flasks; transferring the contents of the second flasks to afed-batch fermentor containing a fermentation composition; fermentingthe contents of the fed-batch fermentor with a fermentation composition,wherein at least one fermentation composition is of claim
 4. 19. Amethod of fermenting Neisseria, comprising: inoculating a vial ofNeisseria to a first flask containing a fermentation composition;cultivating the Neisseria in the first flask; transferring the Neisseriafrom the first flask to a plurality of second flasks containing afermentation composition; fermenting the contents of the second flasks;transferring the contents of the second flasks to a fed-batch fermentorcontaining a fermentation composition; fermenting the contents of thefed-batch fermentor with a fermentation composition, wherein at leastone fermentation composition is of claim
 7. 20. A method of producingNeisseria polysaccharide, the method comprising: fermenting Neisseria ina fermentation composition wherein the fermentation composition does notcomprise NH₄Cl, and harvesting Neisseria polysaccharide.